Extraction of 4-amino-3-isoxazolidone



Patented July 29, 1958 EXTRACTION OF 4-AMINO-3-ISOXAZOLIDONE Eugene E.Howe, Bound Brook, N. J., assignor to Merck & Co., Inc., Rahway, N. J.,a corporation of New Jersey No Drawing. Application February 4, 1955Serial No. 486,276

4 Claims. (Cl. 260-307) This invention relates to novel processes forthe purification of antibiotics; more particularly, it is concerned withmethods of purifying D-4-amino-3-isoxazolidone and obtaining thisproduct in pure crystalline form.

It has recently been found that the growth of the microorganism known asStreptomyces garyphalus in suitable fermentation mediums produces avaluable antibiotic, D-4-amino-3-isoxazolidone, also known as Antibiotic106-7, a potent therapeutic agent active against both gram-negative andgram-positive organisms.

This antibiotic and methods of preparing the same by fermentation aredescribed in detail in the application of Dale A. Harris, Frank J. Wolfand Robert L. Peck, Serial No. 365,271, filed June 30, 1953.

D-4-amino-3-isoxazolidone is an amphoteric substance with both acidicand basic characteristics. Thus, this antibiotic forms salts by reactionwith bases. For example, by reacting this product with bases such assodium hydroxide, potassium carbonate, calcium hydroxide and magnesiumhydroxide, the sodium, potassium, calcium, and magnesium salts ofD-4-amino-3-isoxazolidone are formed. Acid addition salts of thisantibiotic such as the hydrochloride, sulfate, and the like are preparedby adding the appropriate acid to an aqueous solution of the antibioticbase.

D-4-amino-3-isoxazolidone has the structural formula and ischaracterized and identified by the following properties:

(1) Being effective in inhibiting the growth of both gram-positive andgram-negative organisms;

(2) Being soluble in water and insoluble in anhydrous alcohols, acetone,chloroform, ethyl acetate, and pyridine;

(3) Decomposing on heating at a temperature of about 150-155 C.;

(4) Exhibiting in aqueous solution at a pH of about 6 an ultra-violetabsorption maxima at about 2260 A.;

And exhibiting in anhydrous crystalline form absorption bands in theinfra-red region of the spectrum when suspended in solid form in ahydrocarbon oil at the following frequencies, expressed in microns2.8-4.4 (multiple bands), 4.65, 6.12, 6.20 (shoulder), 6.30, 6.42, 6.52,7.10, 7.3, 7.5, 7.85, 8.15, 8.55 (shoulder), 8.80, 9.40, 10.68, 10.9,11.2-11.3 (shoulder), 11.38, 12.08, 13.25 and 15.

The isolation of D-4-amino-isoxazolidone and its purification have beenparticularly difficult since this product does not respond to the usualmethods employed in the isolation and purification of antibiotics, suchas adsorption on charcoal or alumina, or extraction with common solventssuch as ether and butanol. One method of isolating and efiecting asubstantial purification of this antibiotic is described in thecopending application of Rudolf P. Buhs, Edward J. Newstead and NelsonR. Trenner, Serial No. 298,844, filed July 14, 1952, now abandoned. Thisprocedure involves treating an aqueous solution ofD-4-amino-3-isoxazolidone with a strong ly acidic cation-exchange resinto adsorb the antibiotic,

and then cluting the antibiotic from the resin adsorbate with an aqueoussolution of a base. From the basic eluate so obtained, it is possible torecover a product of greatly enhanced potency having up to about 30times the activity of the starting material. Another method of furtherpurifying D-4-amino-3-isoxazolidone is described in the copendingapplication of Irving Putter, Serial No. 379,280, filed September 9,1953, now abandoned. This method comprises adsorbing Antibiotic 106-7onto a strongly basic quaternary ammonium type of anion exchange resinderiving its capacity from quaternary amine groups present in thepolymer, and eluting the product with a solution containing a source ofanions, such as an inorganic acid, an inorganic base, or an inorganicsalt. For example, upon adsorbing D-4- amino-3-isoxazolidone from anaqueous solution having a pH of about 8.5 and assaying about 800 u./ml.obtained by the method shown in copending application Serial No.298,844, onto a strongly basic anion exchanger of the quaternaryammonium type known as Amberlite IRA-400 on the hydroxyl cycle, andeluting the product with a 2% aqueous solution of acetic acid, an eluateassaying about 3-4000 u./ml. can be obtained. However,D-4-amino-3-isoxazolidone recovered from such purified concentrates mustbe purified still further to obtain a substantially pure crystallineproduct.

It is an object of the present invention to provide a process forefiecting the further purification of D-4- amino-3-isoxazolidone andobtaining the product in substantially pure crystalline form. Otherobjects will be apparent from the detailed description of my inventionhereinafter provided.

In accordance with this invention, it is now found that substantialpurification of D-4-amino-3-isoxazolidone is achieved by extracting aconcentrated aqueous solution of this product at an alkaline pH with alower aliphatic alcohol, and recovering the product from the resultingalcohol extracts. Thus, by this method of selectively extracting anaqueous solution of a salt formed by reacting D-4-amino-3-isoxazolidonewith a base with a lower slower aliphatic alcohol, it is possible toefiect a considerable purification of the product. For example, byacidifying the alcoholic extract to a pH of about 5.5 to 6 and allowingthe antibiotic to crystallize from solution, it is possible to recovercrystalline D-4-amino-3-isoxazolidone having an activity of 300-325units per milligram.

Alternatively, the purified product can be recovered from the alcoholicextracts by evaporating the solvent or by precipitation in the form ofan alcohol insoluble salt, such as the sulfate.

In carrying out the processes of my invention, the aqueous solution ofD-4-a1nino-3-isoxazoliclone used as the starting material may be madealkaline with any of the common Water-soluble bases such as an alkalimetal hydroxide, an alkaline earth metal hydroxide, or ammoniumhydroxide. Alternatively, any of the water solublc organic bases such asthe lower aliphatic amines, including quaternary amines, may also beused. Although any of these various bases may be employed in my process,I generally prefer to utilize an alkali metal base, such as sodium orpotassium hydroxide, or ammonium hydroxide since the use of these basesresults in the obtainment of maximum yields of the antibiotic underoptimum conditions. The pH of the aqueous concentrate being extractedmay be from about 8 to 12 or above, although generally I find a pH of9-10 to be most satisfactory.

Any of the lower aliphatic alcohols such as methanol, ethanol, andisopropanol, or mixtures of such alcohols, can be used as the extractingsolvent in my process. The use of a mixture of ethanol and isopropanolis particularly advantageous in my process since it results in theobtainment of substantially pure crystalline product in high yields.

The alcoholic extracts containing the salt of D-4-amino- 3-isoxazolidoneextracted from the aqueous concentrate is adjusted to a pH of about 5.5to 6.0 which is the natural pH of D-4-amino-3-isoxazolidone base; i. e.its zwitter-ion form. Both inorganic acids such as bydrochloric orsulfuric acid, and organic acids such as carboxylic acids, and sulfonicacids can be used to acidify the alcoholic extract. Generally, I find itmost advantageous to use a lower aliphatic acid such as acetic acid orpropionic acid for this purpose since the sodium and potassium salts ofsuch acids are soluble in the alcoholic solvent and do not precipitatewith the crystalline antibiotic. Additionally, the use of such acids isdesirable since the salts formed operate as buffers to maintain thealcoholic solution at the desired pH.

My method of purifying D4-amino-3-isoxazolidone and obtaining thisproduct in crystalline form is particularly useful when employed inconjunction with the process described and claimed in Serial No.379,280, filed September 9, 1953. Thus, for example, an eluate obtainedby eluting the antibiotic from the strongly basic anion-exchangeadsorbate with an aqueous solution of acetic acid, is first made basicto a pH of about 9.5 by the addition of potassium hydroxide, and thevolume of the resulting solution reduced by evaporation under diminishedpressure until the concentrate contains about 20,00040,000 units/mg. ofD-4-amino-3-isoxazolidone. To this resulting concentrate containing thepotassium salt of D-4-amino-3-isoxazolidone is added about 10 volumes ofa 1:1 mixture of ethanol and isopropanol. The resulting clarifiedalcoholic extract containing the potassium salt of the antibiotic isthen acidified with glacial acetic acid to a pH of about 5.7-6.0 andcooled with stirring for several hours thereby causing the antibiotic tocrystallize. The crystallized D-4-amino-3- isoxazolidone so obtainedusually exhibits a potency of about 275-325 units per milligramdepending upon the purity of the starting material.

The following examples are presented for the purpose of illustrating mymethod of purifying D-4-amino-3- isoxazolidone and providing a betterunderstanding of my invention.

Example 1 The rich eluate obtained by the purification method of SerialNo. 298,844 described below was adjusted to pH 10 with sodium hydroxide.The alkalized rich eluate was then concentrated to 350 ml. withcontinual addition of sodium hydroxide suflicient to maintain the pH atabout 10.0. The concentrated solution assayed 22.000 Staph. aureusunits/ml. Ten milliliters of this concentrate were diluted with aboutfifty ml. of isopropanol. The isopropanol insoluble oily layer wasseparated and washed with a small volume of isopropanol. The isopropanolextracts were combined and acidified to about pH 6.0 with glacial aceticacid and the resulting solution cooled in an ice bath for one hour. Thecrystals of D-4-amino-isoxazolidone obtained weighed 300 mg. and assayedabout 250 units/mg.

The rich eluate used as the starting material in this example, wasobtained by eluting D-4-amino-3-isoxazolidone with 0.3 N ammoniumhydroxide solution from an Amberlite IR-120 resin adsorbate prepared bycontacting this resin on the sodium cycle with a solution of 100 g. ofD-4-amino-3-isoxazolidone sulfate (potency 60-88 u./mg.) in four litersof water.

4 Example 2 Three-hundred ml. of the alkaline solution of D4-amino-3-isoxazolidone concentrate prepared as described in Example 1(22,000 units/ml.) was diluted with 1500 ml. of isopropanol. Theresulting solution was centrifuged and filtered with 3 grams ofSupercel. The filtrate was cooled in an ice bath to about 0 C., adjustedto about pH 6.0 with glacial acetic acid, and stirred for one hour afterseeding with crystalline D-4-amino-3- isoxazolidone. The crystals formedwere filtered, washed with isopropanol, ether, and dried under reducedpressure. The dry weight of'the crystalline antibotic was 5.5 grams andassayed about 290 units/ mg.

The isopropanol mother liquor was diluted with water to 100 ml. andtreated with 800 m1. ethanol. Four grams of Supercel was added and themixture filtered. The filtrate was cooled to about 0 C. in an ice bathand acidified to pH 6.0 with glacial acetic acid. The clear supernatantliquor was decanted from the oil which formed and stirring continued forone hour. The crystals 4 obtained from the supernatant were filtered,washed with again diluted with five volumes of isopropanol.

ethanol and ether, and dried in vacuo. The Weight of crystals obtainedwas 6.2 grams with a potency of about 260 units/mg.

Example 3 Thirty-four thousand ml. of resin eluate (assay=l670 units) ofD-4-amino-3-isoxazolidone prepared as described below was treated with3,000 g. of activated charcoal at pH 7.5. The charcoal was removed byfiltration and the filtrate concentrated to about 3650 ml. Theconcentrate was then diluted with about five volumes of isopropanol andthe isopropanol layer decanted from the aqueous oil. The oily phase wasdiluted to 500 ml. with water and The isopropanol extracts werecombined, stirred with grams of Supercel and filtered. The D-4-amino-3-isoxazolidone was crystallized by stirring the filtrate in an ice bathand adjusting the pH to 6 with glacial acetic acid. The crystallineproduct weighed about 53 grams and had a potency of 240 units/mg.

One gram of the crystals so produced was dissolved in about 10 ml. ofwater and adjusted to pH 12 with potassium hydroxide. The solution wastreated with 20 ml. of ethanol and 40 ml. of isopropanol. A small amountof Supercel was added and the mixture filtered. The filtrate was cooledin an ice bath and adjusted to pH 5.8 with glacial acetic acid. Thecrystals obtained weighed 710 mg. and had a potency of about 300units/mg.

The resin eluate employed as the starting material in this example wasobtained by eluting D-4-amino-3- isoxazolidone adsorbed in AmberliteXE-98 with a 2% solution of acetic acid.

Example 4 Fifteen ml. of a concentrated Amberlite XE-98 eluate preparedas shown in Example 3, adjusted to pH 9.0 with potassium hydroxide andcontaining 44,000 units of D-4-amino-3-isoxazolidone was then treatedwith 75 ml. of ethanol and 75 ml. of isopropanol. 1.5 grams of Supercelwas added and the resulting slurry filtered. The filtrate was cooled inan ice bath and acidified with glacial acetic acid to pH 6.0. Thecrystalline product thus obtaned weighed 1.2 grams and had a potency ofabout 310 units/mg.

Example 5 To ten ml. of an Amberlite XE-98 eluate prepared as shown inExample 3 containing 18,000 units of D-4- amino-S-isoxazolidone madealkaline with potassium hydroxide, was added 50 m1. of ethanol and 50ml. of isopropanol. The solution was agitated for about five minutes atroom temperature and centrifuged to remove the oily layer (about 10 ml.)which separated. The alcoholic'extract was cooled to about 5 C. andglacial acetic acid added until the pH dropped to about 6.0. The mixturewas stirred at 0-5 C. for about one hour. The crystals were collected byfiltration, washed with isopropanol and ether and dried in vacuo at 25C. The product weighed 300 mg. and assayed 295 units/mg.

Example 6 About one liter of an aqueous solution containing about3,210,000 units of D-4-amino-3-isoxazolidone was alkalized to a pH of8.5, treated with activated charcoal and filtered. The pH of thefiltrate was adjusted to about 10.5 to 11.0 and concentrated underreduced pressure to a final volume of 100 ml. The concentrate wasdiluted with 500 ml. of isopropanol and 500 ml. of ethanol. Severalgrams of Supercel was added and the mixture stirred and filtered. The pHof the alcoholic extract (filtrate) was then adjusted to about 5.8 andcooled. The crystalline D-4-amino-3-isoxazolidone thus produced had apotency of about 305 units/mg.

D-4-amino-3-isoxazolidone and its acid salts and metal salts are usefulantimicrobial agents. Thus, they can be utilized to remove susceptiblemicroorganisms from pharmaceutical equipment and the like, or toseparate certain microorganisms from solutions containing mixtures ofseveral microorganisms. In addition, they are useful for topicalapplication and aid in restoring healthy tissue. For this purpose theycan be used in solution, or in the form of an ointment and the likeprepared in accordance with procedures known in the art.

Further, D-4-amino-3-isoxazolidone and its acid and metal salts areuseful growth promoting agents for certain animals. Thus, swine whichwere fed an adequate diet supplemented with small amounts ofD-4-amino-3-isoxazolidone gained weight more rapidly than thosemaintained on the unsupplemented diet.

Also, D-4-amino-3-isoxazolidone or its salts are useful in the treatmentof animals infected with microorganisms which are susceptible to theaction of these products. For example, it has been found that they areeffective in the treatment of bovine mastitis.

The D-4-amino-3-isoxazolidone or salts thereof can be used either in theform of pure products or concentrates thereof prepared as describedabove depending upon the particular purpose for which it is intended.Usually, it is preferred to use the antibiotic agent in the form of ametal salt since these salts are unusually stable.

D-4-amino-3-isoxazolidone is also known by the nam oxamycin.

This application is a continuation-impart of Serial No. 386,391 filedOctober 15, 1953, now abandoned.

Various changes and modifications in the procedures herein disclosedwill occur to those skilled in the art, and to the extent that suchchanges and modifications are embraced by the appended claims, it is tobe understood that they constitute part of my invention.

I claim:

1. A process which comprises intimately contacting an aqueous solutionof an alkali metal salt of D-4-amino-3- isoxazolidone, said solutionhaving a pH of about 8 to 12 and containing D-4-amino-3-isoxazolidone ina concentration in excess of about 15,000 units/ml., with at least fivevolumes of an alcohol from the group consisting of ethanol andisopropanol, separating the alcoholic extract, acidifying said alcoholicextract to a pH of about 5.5 to 6.0 with acetic acid, and recoveringcrystalline D-4-amino-3-isoxazolidone from the resulting acidifiedalcoholic extract.

2. The process of claim 1 wherein the extracting alcohol is a mixture ofethanol and isopropanol.

3. The process of claim 1 wherein the alkali metal salt is the sodiumsalt.

4. The process of claim 1 wherein the alkali metal salt is the potassiumsalt.

References Cited in the file of this patent UNITED STATES PATENTS OTHERREFERENCES Gattermann: Organic Chemistry (Macmillan), pp. 43-44 (1923).

MacArdle: Use of Solvents in Synthetic Org. Chem. (Van Nostrand), pp..40, 74 (1925).

1. A PROCESS WHICH COMPRISES INTIMATELY CONTACTING AN AQUEOUS SOLUTIONOF AN ALKALI METAL SALT OF D-4-AMINO-3ISOXAZOLIDONE, SAID SOLUTIONHAVING A PH OF ABOUT 8 TO 12 AND CONTAINING D-4-AMINO-3-ISOXAZOLIDONE INA CONCENTRATION IN EXCESS OF ABOUT 15,000 UNITS/ML., WITH AT LEAST FIVEVOLUMES OF AN ALCOHOL FROM THE GROUP CONSISTING OF ETHANOL ANDISOPROPANOL, SEPARATING THE ALCOHOLIC EXTRACT, ACIDIFYING SAID ALCOHOLICEXTRACT TO A PH OF ABOUT 5.5 TO 6.0 WITH ACETIC ACID, AND RECOVERINGCRYSTALLINE D-5-AMINO-3-ISOXAZOLIDONE FROM THE RESULTING ACIDIFIEDALCOHOLIC EXTRACT.